14/07/10

Sexual crosses

The following protocol is intented for providing a gentic confromation that a observed phenotype of a constructed deletion strain (using hygromycin resistance) is caused by the introduced mutation (essentially checking for unintented and unmarked secondary mutations in the strain). The cross of the mutant and the tester (here wild type) should show that the observed phenotype is 100% linked to the hygromycin resistance.

However, with small modifications the protocol can also be used for construction of double deletion strains by varying the selection pressure and assay conditions.

Protocol

1. Place mycelial plugs (3-4 mm) of the two strains you want to cross on a thick 9 cm carrot agar plate. One strain should be HygromycinS, the other HygromycinR. Make 5 plates for each cross.

2. Incubate at 20 ^oC under (ca. 25 cm) a mixture of continuous cool white and black-light fluorescent lighting. To secure high humidity the plates are incubated in a clear plastic box with a lid, and a beaker of water is placed inside.

3. After approx. 7 days of growth, when mycelia meet at the middle, apply 2 ml of 2.5% aqueous Tween 60 solution and spread with a Drigalsky spatula to flatten the mycelium.

       Incubate for an additional 10 – 14 days.                                    

4. Now perithecia has formed.

5. Pick a perithecium along the line between the two strains with a pair of forceps.

Transfer the perithecium to an Eppendorf tube with 400 µl sterile MilliQ water and squeeze the perithecium using the pair of forceps to release the ascospores from the perithecium.

Vortex briefly and transfer 20 µl spore suspension to a microscope slide to check the ascospore quality in a microscope.

6. Add 600 µl sterile MilliQ water to the spore suspension and filter through a sterile pasteur pipette with a bit of glass wool. The glass wool will hold back any hyphae but let ascospores pass.

7. Divide the spore suspension outcome in two Eppendorf tubes. Plate 20 µl portions from one tube on 14 cm PDA agar plates and from the other tube on 9 cm PDA hyg₇₅ agar plates. Spread with a Drigalsky spatula.

Incubate at 20 ^oC for 3 days to allow spore germination.

8. Count colonies on the PDA+/- hyg to determine if it is a cross.

9. Draw a grid of 6 x 6 lines on 9 cm PDA+/- hyg₁₅₀ agar plates.

Transfer all colonies from the 14 cm PDA agar plates to PDA and PDA hyg₁₅₀  agar plates using s sterile toothpick. There is room for 34 colonies on each plate.

Incubate at 20 ^oC for 4 days.

10. Score progeny for Hygromycin resistance and colour variation.

PDA                                                    PDAhyg₇₅               After 2 days 

PDA                                                     PDAhyg₇₅            After 5 days

Preparing the carrot-agar  (Klittitch 1988)

1. Chop 400 g of baby carrots in a food processor

2. Place the carrots in a 2 L glass beaker, add 400 ml deionised water, cover loosely with Saran wrap and microwave 2 times at 10 minutes

3. Let it cool for 1 to 2 hrs

4. Puree in a food processor

5. Return to beaker and add 500 ml water, mix well

6. Dispense evenly into 5 x 500 ml Pyrex bottles (ap. 225 ml per bottle) containing 4 g agar per bottle (ap. 1.8 % agar)

7. Autoclave for 20 minutes.

Tween 60 solution, 2.5 %

Tween 60 stock is highly viscous; microwave about 20 seconds to liquefy. Using wide-bore pippet, transfer 3.75 ml to a 250 ml Pyrex bottle containing 146.5 ml warmed deionised water. Autoclave.